The present invention generally provides a method of measuring the biological activity of Coagulation Factor VIIa (Factor VIIa). Specifically, the present invention provides a method for directly measuring the activity of Factor VIIa in a plasma sample. More specifically, the present invention provides a method for utilizing Factor VIIa activity as a bio-marker to monitor Factor VIIa inhibition to screen compounds which are able to inhibit the biological activity of Factor VIIa.
In the coagulation cascade, Factor VII plays an important role in clot formation. Factor VII is activated by tissue factor (TF) to form Factor VIIa. As one of its functions, Factor VIIa catalyzes the activation of Factor X to Factor Xa which leads to clot formation.
The TF/VIIa complex is thought to play a key role in thrombogenesis in patients with acute coronary syndromes. Therefore, Factor VIIa is an attractive target for antithrombotic therapy. Because Factor VIIa inhibitors are attractive therapeutic agents, a number of such compounds are under scientific evaluation in clinical development.
Methods for detecting the bioactivity of Factor VIIa inhibitors in plasma are essential for identifying and developing Factor VIIa inhibitors. Prior art methods for measuring Factor VIIa in plasma include the use of truncated tissue factor agents as disclosed in U.S. Pat. Nos. 5,472,850; 5,741,658 or indirect, 2-stage assays which are based on the conversion of Factor X to Factor Xa as catalyzed by Factor VII to provide an indirect measure of Factor VIIa activity and are disclosed in the COASET(copyright) method (Chromogenix AB, Mxc3x6lndal, Sweden). Since the COASET(copyright) method involves the cleavage of a Factor Xa chromogenic substrate following the activation of Factor X, this method does not provide a direct measure of Factor VIIa activity.
Accordingly, it would be both desirable and advantageous to have a plasma-based, direct (1-step) Factor VIa activity assay which can be utilized as an ex vivo plasma bio-marker assay for obtaining accurate Factor VII bioactivity measurements and also for use in screening compounds for their ability to inhibit Factor VIIa in vivo.
The present invention provides a method for directly measuring the activity of Coagulation Factor VIIa (Factor VIIa) in a plasma sample. The method comprises the step of combining a quantity of a plasma sample with a solution comprising a buffer, Factor VII, and a detectable Factor VIIa substrate, and then detecting the amount of the detectable substrate catalyzed to determine the activity of Factor VIIa in the sample.
The present invention also discloses a method for determining whether a compound is useful for inhibiting Factor VIIa biological activity.